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AbstractArctigenin, a phytochemical derived from the Asteraceae family, specifically from
Apoptosis, a form of programmed cell death, is crucial for maintaining cellular homeostasis and is involved in various physiological processes including cell turnover, immune system functionality, and embryonic development. Dysregulation of apoptosis can lead to numerous diseases, including developmental disorders, autoimmune diseases, neurodegeneration, and cancer (10). A complex network of proteins regulates apoptosis and can be classified into two main pathways: the intrinsic (mitochondrial) pathway and the extrinsic (death receptor) pathway. Apoptosis is tightly regulated due to its irreversible nature once initiated.
The control of apoptosis in the intrinsic pathway is mediated by the Bcl-2 protein family, which is categorized into three classes: anti-apoptotic proteins (e.g., Bcl-2, Bcl-xL, Mcl-1), pro-apoptotic proteins (e.g., Bax, Bak), and BH3-only proteins (e.g., Bad, Bik, Bid, Bim, Bok). The Bax/Bcl-2 ratio serves as a critical determinant of a cell’s susceptibility to apoptosis, with lower ratios potentially conferring resistance to apoptosis. This ratio also influences tumor progression and aggressiveness (11).
Inhibitors of apoptosis proteins (IAPs) are classified into three groups: I (e.g., X-linked IAP, XIAP), II, and III. XIAP, a prominent member of class I IAPs, is a potent inhibitor of caspase activity, specifically binding to and inhibiting caspases 3, 7, and 9. Survivin, a member of the smallest class III IAPs, inhibits caspase 9 activity by interacting with upstream regulators involved in mitochondrial-dependent apoptosis (12,13).
Breast cancer is the most prevalent cancer and the second leading cause of cancer-related deaths among women globally, following lung cancer (14,15). It is a complex and heterogeneous disease, typically classified based on receptor status into three categories: estrogen receptor-positive (ER+), human epidermal growth factor receptor 2-overexpressing (HER2+), or triple-negative breast cancer (TNBC), characterized by the absence of estrogen, HER2, and progesterone receptors (16). Receptor status is crucial for determining treatment strategies, such as tamoxifen for ER+ cancers or trastuzumab for HER2+ cancers. While ER+ cancers, which are dependent on estrogen for growth, generally have a better prognosis with estrogen-reducing therapies, HER2+ cancers have seen significant improvement in prognosis due to targeted treatments like trastuzumab (17). In contrast, TNBCs, lacking targeted therapies, have a poorer prognosis and are often associated with drug resistance and metastatic spread, highlighting the need for new therapeutic agents.
Thus, this study evaluates the cytotoxicity of arctigenin against ER–/HER2+/PR– type (SK-BR-3) or TNBC, that is to say, ER–/HER2–/PR– type (MDA-MB-231) cells and investigates its effects on apoptosis in these cancer cells.
Actigenin used in this study was purchased from Sigma-Aldrich. Actigenin was dissolved using dimethyl sulfoxide and then diluted with experimental media in the experiment.
The AGS gastric adenocarcinoma cells (American type cell collection CRT-1739) and breast cancer cell lines MCF-7, SK-BR-3, and MDA-MB-231 were obtained from the Korean Cell Line Bank. AGS cells were cultured in DMEM supplemented with 10% fetal bovine serum and streptomycin-penicillin (100 μg/mL and 100 IU/mL). MCF-7, SK-BR-3, MDA-MB-231, and cells were maintained in RPMI 1,640 medium with 10% fetal bovine serum and streptomycin-penicillin (100 μg/mL and 100 IU/mL). All cell lines were incubated at 37°C in a humidified atmosphere with 5% CO2.
Cells were seeded into 96-well plates at a density of 1 × 104 cells per well. After 24 hours, the cells were treated with various concentrations of arctigenin (0–50 μM) and incubated for an additional 24 hours. Cell viability was assessed using the EZ-Cytox cell viability assay kit (DoGenBio) according to the manufacturer’s instructions. Ten microliters of WST solution were added to the culture medium, followed by a 2-hour incubation in the CO2 incubator. The Absorbance was measured at 450 nm using a spectrophotometer.
Apoptosis was detected using the Annexin V-FITC Apoptosis Detection Kit I according to the manufacturer’s protocol. Cells were trypsinized, washed twice with cold phosphate beffered saline (PBS), and centrifuged at 1,763 g for 5 minutes. The pellet was resuspended in 500 μL of binding buffer at a concentration of 5 × 105 cells/mL, and 100 μL of the cell suspension was transferred to a new 5 mL tube. Then, 5 μL of Annexin V-FITC and 5 μL of propidium iodide (PI) were added. The mixture was incubated for 10 minutes at 37°C in the dark. Following incubation, 400 μL of binding buffer was added, and the samples were analyzed by flow cytometry.
The cells were washed with PBS and lysed at 4°C with a buffer containing 1% Triton X-100, a protease inhibitor cocktail, and PBS. The lysates were centrifuged at 7,605 g for 10 minutes at 4°C, and the supernatants were collected. Protein concentrations were determined using the Lowry protein assay. Proteins were separated by SDS-PAGE for 90 minutes at 120 volts and transferred to a nitrocellulose membrane for 90 minutes at 400 mA. The membrane was incubated overnight at 4°C with primary antibodies at optimal concentrations, followed by a 1-hour incubation with the appropriate secondary antibodies at room temperature. The antibodies used are listed in Table 1, with GAPDH serving as an internal control. Immunoreactive proteins were visualized using enhanced chemiluminescence solution.
List of antibodies used in the present study
| Name of antibody | Species | Dilution ratio | Molecular weight (kDa) | Supplier (Catalog No.) |
|---|---|---|---|---|
| PARP | Rabbit | 1:1,000 | 89, 116 | Cell signaling (9496) |
| Cleaved caspase 3 | Rabbit | 1:3,000 | 17, 19, 32 | Cell signaling (9661) |
| Cleaved caspase 8 | Rabbit | 1:3,000 | 18, 41, 43 | Abcam (ab227430) |
| Cleaved caspase 9 | Rabbit | 1:3,000 | 35, 37, 47 | Cell signaling (9502) |
| Bax | Rabbit | 1:3,000 | 20 | Cell signaling (2772) |
| Bcl2 | Mouse | 1:1,000 | 26 | Cell signaling (4223) |
| XIAP | Rabbit | 1:3,000 | 53 | Cell signaling (2045) |
| Survivin | Rabbit | 1:1,000 | 16 | Cell signaling (2808) |
| GAPDH | Mouse | 1:1,000 | 37 | Santa Cruz (sc-47724) |
Data in the bar graphs are presented as mean ± standard error of the mean. Statistical analyses were conducted using GraphPad Prism 5.02 software (GraphPad Software). Data were analyzed by unpaired Student’s
To investigate its potential impact on gastric cancer, we assessed the effect of arctigenin on AGS cells, a gastric adenocarcinoma cell line. AGS cells were treated with arctigenin at concentrations ranging from 0.39 μM to 50 μM for 24 hours, and cell viability was measured using the WST assay. No significant changes in cell viability were observed in AGS cells (Fig. 1A). In addition, we evaluated the effect of arctigenin on the viability of various breast cancer cell lines: MCF-7 (ER+/HER2–/PR–), SK-BR-3 (ER–/HER2+/PR–), and MDA-MB-231 (ER–/HER2–/PR–) cells. Cells were treated with arctigenin at concentrations between 0.39 μM and 50 μM for 24 hours, followed by the WST assay. ER+ MCF-7 cells exhibited almost no significant effects at lower concentrations than 50 μM of arctigenin (Fig. 1B). In contrast, the viability of ER– SK-BR-3 and ER– MDA-MB-231 cells decreased as arctigenin concentrations increased, with SK-BR-3 cell viability reduced by 62.1% and MDA-MB-231 cell viability reduced by 59.4% at 6.25 μM arctigenin (Fig. 1C, 1D).
Fig. 1. Effect of arctigenin on the viability of cancer cell lines. Cells were treated with an indicated dose of arctigenin for 24 hours and cell viability was measured by WST assay. (A) AGS. (B) MCF-7 (ER+, PR–, HER2–). (C) MDA-MB-231 (ER–, PR–, HER2–). (D) SK-BR-3 (ER–, PR–, HER2+). Results from duplicate experiments were analyzed by Student’s To determine whether arctigenin induces apoptosis, SK-BR-3 cells were treated with 0, 125, 250, and 500 nM arctigenin. Apoptosis was assessed using Annexin V-FITC/PI staining and flow cytometry, and PARP cleavage was evaluated by Western blot analysis. Flow cytometry revealed that the percentage of apoptotic cells increased from 4.14% to 6.34%, 7.92%, and 8.20% at 0, 125, 250, and 500 nM arctigenin, respectively (Fig. 2A, 2B). Western blot analysis demonstrated a decrease in full-length PARP and an increase in cleaved PARP with higher arctigenin concentrations (Fig. 2C, 2D). These findings indicate that arctigenin induces apoptosis in SK-BR-3 cells in a dose-dependent manner.
Fig. 2. Arctigenin induces apoptosis in SK-BR-3 cells. (A) SK-BR-3 cells were treated with an indicated dose of arctigenin for 24 hours, then stained with annexin V-FITC and PI, and subjected to flow cytometry. Stained cells were analyzed and illustrated on the quadrant by CellQuestPro software. (B) The percentage of apoptotic or live cells from flow cytometry results was analyzed and illustrated as a graph. (C) The cell lysates were subjected to Western blot to evaluate the levels of PARP, cleaved PARP, caspase 3, cleaved caspase 3, caspase 8, cleaved caspase 8, caspase 9, cleaved caspase 9, Survivin, and XIAP. GAPDH was used as an internal control. (D) Densities of the Western blot bands were illustrated as a graph and the results from triplicate experiments were analyzed by Student’s To further elucidate the mechanism of apoptosis induction by arctigenin, we analyzed the expression of caspases in SK-BR-3 cells treated with arctigenin. Western blot analysis showed a reduction in the levels of full-length caspases 3, 8, and 9, accompanied by an increase in their cleaved forms (Fig. 2C, 2D). These results suggest that arctigenin induces apoptosis in SK-BR-3 cells through a caspase-dependent mechanism involving both intrinsic (via caspase-9) and extrinsic (via caspase-8) pathways.
IAPs, such as XIAP and Survivin, inhibit apoptosis by directly binding to and inhibiting caspases, including caspases 3, 7, and 9. The expression levels of XIAP and Survivin in SK-BR-3 cells treated with arctigenin were assessed using Western blot analysis. The results indicate that arctigenin treatment led to a reduction in the levels of XIAP and Survivin, with a pronounced decrease observed at 500 nM arctigenin (Fig. 2C, 2D).
The Bcl-2 protein family regulates the intrinsic pathway of apoptosis, with Bax promoting and Bcl-2 inhibiting apoptosis. To evaluate the effect of arctigenin on these proteins, we measured the expression of Bax (pro-apoptotic) and Bcl-2 (anti-apoptotic) in SK-BR-3 cells by Western blot analysis and calculated the Bax/Bcl-2 ratio. Arctigenin treatment resulted in increased Bax expression and decreased Bcl-2 expression (Fig. 3A). The Bax/Bcl-2 ratio increased in a dose-dependent manner, reaching 4.7, 8.7, and 9.6 times that of untreated cells following treatment with 125, 250, and 500 nM arctigenin, respectively (Fig. 3B). These findings suggest that arctigenin-induced apoptosis is associated with the suppression of anti-apoptotic proteins (XIAP, Survivin, and Bcl-2) and the upregulation of the pro-apoptotic protein Bax in SK-BR-3 cells.
Fig. 3. Arctigenin increases the Bax/Bcl-2 ratio in SK-BR-3 cells. SK-BR-3 cells were treated with an indicated dose of arctigenin for 24 hours. The cell lysates were subjected to Western blot. (A) The level of Bax and Bcl-2. (B) Each band intensity from the data was normalized to GAPDH and the Bax/Bcl-2 ratio was calculated. Data were presented as mean ± standard error of the mean of three independent experiments and analyzed by Student’s The effect of arctigenin on apoptosis in MDA-MB-231 cells was evaluated by treating cells with 0, 125, 250, and 500 nM arctigenin for 24 hours, followed by flow cytometry using Annexin V-FITC/PI staining and Western blot analysis. Flow cytometry results demonstrated an increase in apoptosis in arctigenin-treated cells, with the apoptosis ratio rising from 7.13% in untreated cells to 8.98%, 10.86%, and 13.36% at 125, 250, and 500 nM arctigenin, respectively (Fig. 4A, 4B). Western blot analysis revealed a decrease in full-length PARP, caspase 3, and caspase 9, alongside an increase in cleaved PARP, cleaved caspase 3, and cleaved caspase 9 (Fig. 4C, 4D). Additionally, XIAP and Survivin levels were reduced following arctigenin treatment (Fig. 4C, 4D). The Bax/Bcl-2 ratio increased 2.2, 3.2, and 4.1 times in arctigenin-treated cells compared to untreated cells (Fig. 5A, 5B). These results indicate that arctigenin induces apoptosis in MDA-MB-231 cells primarily through the intrinsic apoptotic pathway.
Fig. 4. Arctigenin induces apoptosis in MDA-MB-231 cells. (A) MDA-MB-231 cells were treated with an indicated dose of arctigenin for 24 hours, then stained with annexin V-FITC and PI, and subjected to flow cytometry analysis. Stained cells were analyzed and illustrated on the quadrant by CellQuestPro software. (B) The percentage of apoptotic or live cells was analyzed and illustrated as a graph. (C) The cell lysates were subjected to Western blot to evaluate the levels of PARP, cleaved PARP, caspase 3, cleaved caspase 3, caspase 9, cleaved caspase 9, Survivin, and XIAP. GAPDH was used as an internal control. (D) Densities of the Western blot bands were illustrated as a graph and the results from triplicate experiments were analyzed by Student’s 
Fig. 5. Arctigenin increases the Bax/Bcl-2 ratio in MDA-MB-231 cells. MDA-MB-231 cells were treated with an indicated dose of arctigenin for 24 hours. The cell lysates were subjected to Western blot. (A) The level of Bax and Bcl-2. (B) Each band intensity from the data was normalized to GAPDH and the Bax/Bcl-2 ratio was calculated. Data were presented as mean ± standard error of the mean of three independent experiments and analyzed by Student’s In this study, we evaluated the inhibitory effects of arctigenin on three types of breast cancer cell lines: MCF-7 (ER+/HER2–/PR–), SK-BR-3 (ER–/HER2+/PR–) and MDA-MB-231 (ER–/HER2–/PR–). To evaluate the inhibitory effects of arctigenin on ER-negative breast cancer cells, ER-positive MCF-7 cells were used as a control. A gastric adenocarcinoma AGS cells were also used as a control.
Arctigenin significantly reduced the growth of ER-negative breast cancer cells. SK-BR-3 and MDA-MB-231 cells but did not affect AGS and MCF-7 cell viability. Specifically, arctigenin treatment activated caspases 3 and 9, induced PARP cleavage, and decreased the levels of anti-apoptotic proteins XIAP and Survivin in ER– SK-BR-3 and ER– MDA-MB-231 cells. Additionally, arctigenin treatment increased the Bax/Bcl-2 ratio in these cells, which likely contributed to the activation of caspase 9 and subsequent apoptosis through the intrinsic pathway. If apoptosis was induced in AGS or MCF-7 cells (control groups) by arctigenin treatment, the apoptotic extrinsic pathway would have been mainly activated (activation of Cas-7, -8, -3, and PARP). However, as shown in the WST assay results in result 1, AGS and MCF-7, which were used as controls, did not repeatedly show death in the concentration range set for arctigenin treatment. Therefore, we did not perform Western blotting on AGS and MCF-7. WST is a test that measures the color development of formazan reduced by succinate-tetrazolium reductase, a dehydrogenase present in the respiratory chain of mitochondria and active only in living cells.
Cell viability assays using the WST assay demonstrated that arctigenin did not significantly alter the viability of AGS cells at concentrations ranging from 0.39 μM to 50 μM (Fig. 1A). Susanti et al. (18) reported no cytotoxic effects on normal diploid fibroblasts (WI-38), human fibroblasts (KMST-6), normal embryo fibroblasts (OUMS-36), and normal mammary epithelial cells (H184B5F5/M10) under 50 μM of arctigenin treatment.
MCF-7 cells exhibited almost no reduction in viability at 50 μM arctigenin, while SK-BR-3 cells demonstrated a 22% and 62.1% decrease at the same concentrations, and MDA-MB-231 cells showed a 15.7% and 59.4% decrease in viability at 0.39 μM and 6.25 μM, respectively (Fig. 1B-1D). These findings are consistent with Hsieh et al. (19), who observed that arctigenin inhibited MDA-MB-231 cell growth but had minimal impact on MCF-7 cells at 20 μM for 48 hours. Conversely, Maxwell et al. (8) reported that arctigenin induced MCF-7 cell death through autophagy rather than apoptosis or cell cycle arrest, with a 30% reduction in cell viability at 100 μM for 24 hours. In our result, arctigenin showed decreased cell viability as 22% ± 2% at 100 μM for 24 hours (data not shown).
Feng et al. (5) identified IC50 values of 40 μM and 0.79 μM for MCF-7 and MDA-MB-231 cells, respectively, with arctigenin, correlating reduced MDA-MB-231 viability with apoptosis. Combined with previous studies, our results indicate that the inhibitory effect of arctigenin on MCF-7 cells is less pronounced compared to its effect on MDA-MB-231 and SK-BR-3 cells. This differential sensitivity may be due to the presence of ER in MCF-7 cells, which could reduce the inhibitory effects of arctigenin on cell proliferation compared to ER-negative breast cancer cells (19).
Exposure to cytotoxic agents can induce various cellular responses, including necrosis, cell cycle arrest, autophagy, and apoptosis. Previous studies have demonstrated that arctigenin triggers apoptosis and cell cycle arrest in diverse cancer cell lines (5-9). The current study specifically investigated apoptosis induced by arctigenin. Annexin V-FITC/PI staining and increased levels of cleaved PARP, as detected by Western blot analysis, confirmed that arctigenin induces apoptosis in SK-BR-3 and MDA-MB-231 cells (Fig. 2, 4). Furthermore, arctigenin activates caspases 3, 8, and 9 in SK-BR-3 cells (Fig. 2C, 2D) and caspases 3 and 9 in MDA-MB-231 cells (Fig. 4C, 4D). These results suggest that arctigenin induces apoptosis in SK-BR-3 breast cancer cells (ER–/HER2+/PR–) via both extrinsic and intrinsic pathways, while in MDA-MB-231 cells (ER–/HER2–/PR–), arctigenin induces apoptosis primarily through the intrinsic pathway.
Arctigenin treatment resulted in decreased expression of the anti-apoptotic proteins XIAP and Survivin in both SK-BR-3 and MDA-MB-231 cells (Fig. 2, 4). Survivin, which is regulated by the cell cycle and is expressed predominantly during the G2/M phase, is often overexpressed in cancers, correlating with chemotherapy resistance, increased tumor recurrence, and reduced patient survival. Thus, targeting Survivin represents a promising therapeutic approach (12).
The Bcl-2 family proteins are crucial in regulating the intrinsic apoptotic pathway. This family includes both pro-apoptotic proteins, such as Bax, and anti-apoptotic proteins, such as Bcl-2. Bax promotes the release of cytochrome c from the mitochondria, while Bcl-2 inhibits this release by interfering with Bax activity (11). Released cytochrome c binds to caspase-9, thereby initiating the intrinsic apoptosis pathway. The Bax/Bcl-2 ratio is critical in modulating cytochrome c release and mitochondrial membrane permeability. As shown in Fig. 3, 5, arctigenin increased Bax expression and decreased Bcl-2 expression, leading to enhanced mitochondrial membrane permeability, increased cytochrome c release, and activation of the intrinsic apoptotic pathway.
Based on these findings, arctigenin may be beneficial in the management of ER-negative and TNBCs. However, further in vivo studies are necessary to fully assess the anticancer efficacy of arctigenin.
None.
No potential conflict of interest relevant to this article was reported.
This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (No. RS-2023-00253761).
Conceptualization: THY, SHK. Data curation: SHK, HJW. Formal analysis: SHK, HJW. Funding acquisition: SHK. Investigation: THY, HJW. Methodology: THY, HJW. Project administration: HJY, SHK. Resources: HJW, SHK. Software: HJW, THY. Supervision: SHK, HJW. Validation: SHK. Visualization: THY, HJW. Writing – original draft: THY. Writing – review and editing: SHK, HJW.
